One of the major unresolved mysteries in tumor biology is the mechanism by which tumor cells in vivo exist from the cell cycle in a reversible fashion. An implicit assumption through decades of research is that limitation of nutrient supply, either by limited penetration into the cell mass or restricted blood flow to local regions, create a stress microenvironment which induces cells to arrest their cell cycle transit. Currently, neither the nature of the microenvironmental signal(s) inducing quiescence in tumors nor the molecular mechanism(s) by which this induction occurs are known. The ultimate goal of this research is to determine both of these. Our overall hypothesis is: microenvironmental regulation of tumor cell proliferation is mediated by the expression and activity of specific cyclin dependent kinase inhibitors (CK1s). In other words, we propose that cell cycle rest in tumors is not a passive delay caused by depletion of critical nutrients or a general interruption of energy metabolism, but is rather an active process in which microenvironmental signals cause the induction and activation of specific proteins which induce cell cycle delay. Due to well-known mutations of experimental tumors for such mechanistic studies, we propose to use the multicellular tumor spheroid model for the majority of this project. Spheroids are ideally suited for such studies, both because of their symmetrical arrangement of microenvironmental and cellular proliferation gradients, and because of our unique ability to experimentally exploit this symmetry. Specifically, we can isolate intact, variable cells from known locations within the spheroid microenvironment for detailed study for the molecular changes associated with cell cycle arrest. In order to provide a link between this in vitro system and the in vivo situation we will determine whether our proposed mechanism is operative in actual numbers. We propose the following four Specific Aims: 1) to determine the molecular basis for cell cycle arrests in multicellular spheroids, 2) to determine if the same molecular mechanisms are operative in tumors in vivo; 3) to identify the microenvironmental signal(s) which induce cell cycle arrest in spheroids; and (4) to determine the interaction between radiation- and microenvironmentally-induced cell cycle arrest. We believe that the proposed studies will identify, for the first time, the proteins responsible for microenvironmental control of cell cycle progression in tumor cells, as well as describe the signals responsible for inducing and/or activation these regulatory proteins. Once the molecular basis for quiescence in the solid tumor is defined, novel strategies can be developed to attack these therapy-resistant tumor cells.